Electroneutral Na+coupled HCO-3 transporters (nNCBTs), in the SLC4 family, mediate - HCO3 uptake and (along with Na-H exchangers, NHEs) play important roles in regulating - the intracellular pH (pHi) of neurons. nNCBTs also contribute to HCO3 secretion by choroid plexus and blood-brain barrier endothelial cells. Thus, nNCBTs are in a position to have profound effects on ion channels and on neurotransmitter receptors and transporters, thereby influencing neuronal excitability and synaptic transmission. Preliminary data show that truncating an autoinhibitory domain (AID) from the cytosolic N terminus (Nt) of nNCBTs increases transport more than 2 fold. The cytoplasmic protein IRBIT-which binds to IP3 receptors and inhibits Ca release-also interacts with the Nt of nNCBTs, nullifying inhibition by the AID. A third protein, the phosphatase CnAB, appears to bind to the Nt of one of the nNCBTs and profoundly inhibit transport. These three players (AID, IRBIT, and CnAB) may be the most powerful known proteins controlling acid-base transport. Certain nNCBTs splice variants may lack the AID or binding domains for IRBIT or CnAB. The major goal of this proposal is to understand the control of nNCBTs by the AID and IRBIT (and also by CnAB), and how these interactions modulate neuronal function. The four aims: (1) Use functional assays in oocytes to elucidate the above interactions. (2) Use binding assays to detect the interactions. (3) Determine the contribution of the nNCBTs to pHi regulation in hippocampal (HC) vs. medullary-raphi (MR) neurons (some of which are chemosensitive), and the effect of pHi changes on electrophysiological parameters. This aim exploits mouse knockouts of the three nNCBTs as well as mice with fluorescent-labeled neurons to aid in identification. (4) To express, solubilize, and characterize several ancestral nNCBT proteins. The proposed work will clarify the molecular mechanism and regulation of the nNCBTs, and the role they play in neuronal function. The research could have important implications for understanding the control of ventilation, SIDS, seizure disorders, and perhaps autism.